Clathrin mediated endocytosis (CME) in the fission yeast critically depends on the connection between the lipid membrane and F-actin. The fission yeast endocytic protein End4 (homologous to Sla2 in budding yeast and HIP1R in human) contains a N-terminal domain that binds to PIP2 on the membrane, and a C-terminal THATCH domain that is postulated to be a binding partner of F-actin . Purified THATCH domain of the budding yeast Sla2, however, shows low affinity to F-actin . We tested if isolated THATCH domain still has low affinity to F-actin , using TEV protease (TEVp)-mediated protein cleaving to separate the THATCH domain from the rest of End4. Our results indicate that the isolated THATCH domain of End4 is unable to bind F-actin independently , consistent with the low affinity of the THATCH domain to F-actin measured from binding assays.
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Cross-regulations of two connected domains form a mechanical circuit for steady force transmission during clathrin-mediated endocytosis.
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Nanobiology Institute, Yale University, West Haven, CT 06516, USA; Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address:
Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin.
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Department of Molecular Biophysics and Biochemistry, Yale University.
Clathrin mediated endocytosis (CME) in the fission yeast critically depends on the connection between the lipid membrane and F-actin. The fission yeast endocytic protein End4 (homologous to Sla2 in budding yeast and HIP1R in human) contains a N-terminal domain that binds to PIP2 on the membrane, and a C-terminal THATCH domain that is postulated to be a binding partner of F-actin . Purified THATCH domain of the budding yeast Sla2, however, shows low affinity to F-actin .
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